Immunoassays are commonly done as rate assays, that is, they are measured as a rate of change over time, for example, the rate of increase or decrease in density of a dye. The dye chemistry can be, for example, one that relies upon an enzyme to catalyze a leuco dye to change into a colored dye. A class of enzymes that is frequently used for this purpose is oxidases, preferably peroxidase, or "POD". Most preferably, the peroxidase is horseradish peroxidase, hereinafter "HRP".
Immunoassays involve the measurement of limited amounts of POD, unlike other assays which can use excess amounts of POD. (Excess amounts of course are relatively insensitive to interferents that imitate or react with the POD.) An immunoassay is usually either "competitive" or "sandwich". In "competitive" assays, a labeled analog of the target analyte to be determined is placed in competition with the analyte for a fixed amount of an appropriate, immobilized antibody which can react with either the target analyte or a target analyte analog. The label on the analog can be appropriately detected in either its "free" or its complexed (that is, reacted) form. Signal level then will tell the user how much target analyte is in the sample being tested.
In the alternative immunoassay format of a "sandwich" immunoassay or immunometric assay, the target analyte is contacted with two or more receptor molecules, e.g., antibodies, which bind to the target analyte at different epitopic sites. One receptor molecule is typically appropriately labeled and the other is either immobilized on a solid substrate, or is capable of being immobilized thereon. The amount of target analyte is directly proportional to the amount of bound complex among the target analyte and the two receptors.
In either of these assays, if HRP is used, it is part of the "label". Hence, immunoassays cannot rely on the presence of excessive amounts of HRP to overcome changes due to the presence of interferents.
It has been known in the art that such peroxidase chemistry, when used in immunoassays, is interfered with by substances such as hemoglobin. That is, the presence of any hemoglobin will cause more or less of a bias. In liquid immunoassays, this is corrected by diluting, usually. Such correction by dilution is not available, however, when using slide test elements. The reason is that it complicates the procedure, adds errors, and further lowers an already low concentration of certain analytes. Since hemoglobin is often present in blood samples in unknown amounts, it has been a long-felt problem prior to this invention to somehow correct for the unpredictable bias created by hemoglobin in such immunoassays. Certainly a correction could be done if the amount of hemoglobin present (if any) were first ascertained, but that is a step most laboratories do not wish to do prior to running the immunoassay.